The HIV-1 genome consists of only 9 genes and thus, depends highly on host cellular factors to replicate. Recent studies, through genome-wide siRNA screens, have identified many possible HIV-1 dependency factors HDFs. However, there lies a large inconsistency between these analyses and the majority of potential hits have not been verified in primary human target cells. Therefore, the goal of this proposal is to validat a recently identified possible HIV-1 Env-mediated host factor, Rab6A GTPase, in primary human monocyte-derived macrophages (MDMs) and define the mechanism by which this protein facilitates HIV-1 infection. Additionally, because Rab6A is ubiquitously expressed in two closely related isoforms, Rab6A and Rab6A', that have similar but distinct roles in the cell, both isoforms will be individually investigated. Similar to all GTPases, Rab6A(A/A') function is dependent on specific accessory proteins that allow cycling between active-GTP bound and inactive-GDP bound forms. We hypothesize that activated Rab6A(A/A') is required for efficient HIV-1 Env-mediated infection of MDMs and activation of Rab6A(A/A') is dependent on Ric1/Rgp1, a Rab6A(A/A')-specific guanine nucleotide-exchange factor, activity. We will utilize a pseudoviral single-round infection assay to define the mechanism by which Rab6A and Rab6A' facilitate HIV-1 replication in primary human macrophages. Our studies may reveal a novel therapeutic pathway to combat HIV-1/AIDS, as well as help dissect the fundamental biological role of Rab6A(A/A') in primary human macrophages.